The function of the central nervous system (CNS) is dependent upon the establishment of synaptic junctions during development and the proper patterning of neuronal connectivity to generate functional circuits. Failure to establish the appropriate synaptic connections can result in severe disorders of the nervous system, including mental retardation, autism, schizophrenia and depression. Thus, it is essential to understand the molecular mechanisms responsible both for assembling the synaptic junction, and for directing the selection of functionally appropriate synaptic partners. Our long-term goal is to understand the cellular and molecular mechanisms responsible for synaptic assembly and selection in the vertebrate CNS. To address these questions, we are using in vivo multiphoton imaging of synaptogenesis in the developing zebrafish CNS, focusing on the roles played by 3-protocadherins. The embryonic zebrafish is an ideal system in which to address these questions, as embryos are transparent, develop externally and rapidly, and have a relatively simple and highly stereotyped CNS. In addition, the recent development of site-specific modification of the genome through the use of engineered zinc-finger nucleases (ZFNs) will enable the efficient production of zebrafish lines that harbor mutations in genes of interest. In this exploratory R03 proposal, we will use the emerging ZFN technology to generate lines of zebrafish carrying lesions in each of the 3-protocadherin genes present in zebrafish, pcdh13 and pcdh23. Having generated and validated these lines, we will also perform an initial phenotypic analysis. As targeted deletion of pcdh3 in mice results in extensive neuronal death throughout the CNS, we will first assess the levels of programmed cell death occurring in the developing nervous system. We will also characterize any effects on the organization of the nervous system, using wholemount immunocytochemistry to label the early scaffold of axon tracts. Finally, we will use both immunocytochemistry and live imaging to identify any defects in the formation or maintenance of synaptic vesicle clusters. The pcdh3 mutant lines produced from this work will be invaluable resources for investigating both the cellular and developmental function of Pcdh3, allowing us to take full advantage of the zebrafish model system. PUBLIC HEALTH RELEVANCE: The formation of synaptic junctions and their patterning into circuits of appropriately connected neuronal populations is essential for the normal development of the central nervous system. Defects in these events can have a profound influence on human behavior, and may underlie numerous disorders of the nervous system, including autism, mental retardation and schizophrenia. The results of our studies will greatly enhance our knowledge of 3-protocadherin function in neural circuit assembly, and will provide a strong foundation for developing novel therapeutic strategies for addressing these disorders. These investigations will also contribute to a firm understanding of the developmental mechanisms responsible for synaptogenesis, which could be essential for creating effective approaches to promote repair after brain or spinal cord injuries.